A Nonsense Mutation in the HOXD13 Gene Underlies Synpolydactyly with Incomplete Penetrance

ORIGINAL ARTICLE

A nonsense mutation in the HOXD13 gene underlies synpolydactyly with incomplete penetrance

Mazen Kurban1, Muhammad Wajid1, Lynn Petukhova1, Yutaka Shimomura1 and Angela M Christiano1,2

Synpolydactyly 1 (SPD1; OMIM 186000), also known as type II syndactyly, is a dominantly inherited limb malformation that is characterized by an increased number of digits. SPD1 is most commonly caused by polyalanine repeat expansions in the coding region of the HOXD13 gene, which are believed to show a dominant-negative effect. In addition, missense and out-of-frame deletion mutations in the HOXD13 gene are also known to cause SPD, and the mechanism responsible for the phenotype appears to be haploinsufficiency. Here, we analyzed a large consanguineous family from Pakistan with SPD showing a wide variation in phenotype among affected individuals. We performed genetic linkage analysis, which identified a region on chromosome 2 containing the HOXD13 gene. Haplotype analysis with microsatellite markers suggested segregation of the phenotype with HOXD13 gene with incomplete penetrance. Direct sequencing analysis of HOXD13 gene revealed a nonsense mutation, designated as Q248X. All affected individuals with the severe SPD phenotype are homozygous for the mutation, whereas those with the mild SPD phenotype are heterozygous for the mutation. Furthermore, some unaffected individuals also carry the mutation in the heterozygous state, showing incomplete penetrance. Our results show the first nonsense mutation in the HOXD13 gene underlying a severe form of SPD in the homozygous state, and a milder form of SPD with B50% penetrance in the heterozygous state, most likely because of the production of 50% of protein compared with normal individuals. Journal of Human Genetics (2011) 56, 701–706; doi:10.1038/jhg.2011.84; published online 4 August 2011

Keywords: haploinsufﬁciency; incomplete penetrance; synpolydactyly

INTRODUCTION sequences.7–9 HOXD13 is a member of the HOX family, which Trinucleotide repeat expansions in either the coding or the non-coding encodes for a transcription factor with a crucial role in limb develop- sequences of genes are known to cause several hereditary diseases.1–3 ment. The expression of HOXD13,themost5¢ homeobox gene of the One such example is the polyalanine repeat expansions in HOXD13 HOXD gene cluster, is involved in both the early and late phases of gene, which have been shown to underlie synpolydactyly 1 (SPD1; limb morphogenesis.10–12 The first phase occurs during the emergence OMIM 186000).2 In contrast to other types of nucleotide repeats, such of the limb buds, in which the limbs are endowed with anteroposterior as polyglutamine and polyglutamic acid repeats in Huntington’s disease (AP) polarity via the polarized expression of several genes.11,12 The and Friedreich’s ataxia, respectively, polyalanine repeats in SPD1 are mito- second stage, which is involved in the distal outgrowth and specifica- tically and meiotically stable, and thus polymorphisms tend to be rare.2 tion of the most distal limb regions, depends on sonic hedgehog SPD is a rare autosomal dominant limb deformity, with a distinctive expression.13,14 HOXD genes are critical in both phases, because they combination of syndactyly and polydactyly. The main features of SPD initiate sonic hedgehog expression during the early phase and mediate are webbing of the 3/4 fingers and 4/5 toes, with partial or complete the sonic hedgehog morphogenic signal within the limb during the digital duplication within the syndactylous web.4–6 Currently, SPD is second phase. Therefore, mutations such as repeat expansions in classified into three types, SPD1, SPD2 and SPD3, yet mutations have HOXD13 are expected to result in limb malformation pheno- been identified in only two types. SPD1 is caused mainly by polyalanine types.12,15,16 N-terminal polyalanine repeats of +7 to 14 polyalanine expansion repeats in the HOXD13 gene on chromosome 2.4 SPD2 repeats are the most common mutations described in HOXD13, (OMIM 608180) has been linked to chromosome 22 and mutations in whereas the normal number of polyalanine repeats in HOXD13 is fibulin 1 gene have been reported.4 SPD3 (OMIM 610234) has been 15.12,15,16 To date, only a few missense and deletion mutations have linked to chromosome 14, but no gene has yet been implicated.4 been reported in the HOXD13 gene.17–22 Interestingly, nonsense The HOX family of transcription factors are homeodomain- mutations have not been reported previously in the HOXD13 gene. containing proteins that control cell fates and regional identities In this study, we analyzed a large Pakistani family with SPD1 and along the primary body and limb axes, by binding to cognate DNA identified a first nonsense mutation in HOXD13 gene. Our findings

1Department of Dermatology, College of Physicians and Surgeons, Columbia University, New York, NY, USA and 2Department of Genetics and Development, Columbia University, New York, NY, USA Correspondence: Dr AM Christiano, Department of Dermatology, College of Physicians & and Surgeons, Columbia University, 630 West 168th Street VC-1526, New York, NY 10032, USA. E-mail: [email protected] Received 7 March 2011; revised 9 May 2011; accepted 1 June 2011; published online 4 August 2011 A nonsense mutation in the HOXD13 gene MKurbanet al 702

further underscore the crucial roles of HOXD13 in limb development 3/4/5, camptodactyly of the big toe and brachydactyly, which mainly in humans. affected the second toe (Figure 2). On the other hand, the remaining six affected individuals showed a milder phenotype, which was restricted MATERIALS AND METHODS to the toes and consisted mainly of webbing of the fourth and the fifth Subjects and linkage analysis digits or the second and third digits. In addition, a few affected individuals After obtaining informed consent, we collected peripheral blood samples from also had unilateral broad big toes, which is not a typical feature for SPD1 the family members and 100 population-matched unrelated healthy control (Figure 2). None of the individuals had any urogenital abnormalities. individuals in EDTA-containing tubes (under institutional approval and in adherence to the Declaration of Helsinki Principles). Genomic DNA was Linkage analysis isolated from these samples according to standard techniques. SPD is characterized as an autosomal dominant disorder with wide variability in the phenotypic consequence of particular mutations. Genotyping and data analysis In addition to reduced penetrance, a number of investigators have The Affymetrix GeneChip Human Mapping 10K 2.0 (Affymetrix, Santa Clara, CA, USA) array was used to genotype samples. Sample preparation followed the observed that carriers of two mutations often have a more severe standard Affymetrix protocol, with minor alterations, as reported previously.23 phenotype than heterozygous carriers. We therefore initially per- Hybridization was performed by the Columbia University Gene Chip Facility. formed a parametric linkage analysis under the assumption of a Genespring GT (Agilent Software, Santa Clara, CA, USA) was used for quality dominant mode of transmission with reduced penetrance. This control measures and to perform analyses. Single-nucleotide polymorphisms analysis failed to reveal any regions of linkage. Because of the exten- displaying Mendelian inheritance errors were removed so that the analyzed data sive consanguinity in our pedigree, we considered that a majority set contained 9833 variations. Haplotypes were inferred from the data by using of affected individuals may be homozygous carriers; hence, we Genespring GT to mitigate the effect of linkage disequilibrium on multipoint performed parametric linkage analysis under a recessive mode of linkage analysis, thus reducing type I error. Statistical analyses included non- inheritance. This approach also failed to reveal regions of linkage. parametric linkage analysis, parametric linkage analysis under assumptions of Finally, we performed non-parametric linkage analysis, which does dominant mode of inheritance with reduced penetrance and the haplotype- based haplotype relative risk, which is a family-based test of association. Next not rely on a previous assumption of a mode of inheritance, and genomic DNA from the family members was amplified by PCR using primers discovered a significant non-parametric linkage analysis score at for microsatellite markers around the HOXD13 gene, D2S2188, D2S2314, chromosome 2q22.3–34 (Z¼3.15) (Figure 3). HOXD13-MS, D2S148, D2S2261, D2S2273 and D2S1361. Of these, HOXD13-MS is a highly polymorphic marker, which resides 0.35 Mb down- Haplotype analysis with markers near the HOXD gene cluster stream of the HOXD13 gene, and the following primers were used for the We analyzed a total of 27 members (16 affected and 11 unaffected) of amplification: forward (5¢-CTCAGTGAATGTTCACAATCCA-3¢)andreverse the SPD family. We performed microsatellite marker analysis using (5¢-CAACCAACAAACAAGTGGCTC-3¢). The amplification conditions for seven markers that span the region of linkage. Ten of the 16 affected 1 1 1 each PCR were 94 C for 2 min, followed by 35 cycles of 94 C for 30 s, 55 C individuals were homozygous for a common haplotype, designated for 30 s and 72 1C for 30 s, with a final extension at 72 1Cfor7min.PCR haplotype A, between the markers D2S2188 and D2S148 (Figure 1). products were run on 8% polyacrylamide gels, and genotypes were assigned by visual inspection. This region overlapped with the HOXD13 gene. Six of the affected individuals, as well as six of the 11 unaffected individuals, carried the Mutation analysis of the HOXD13 gene haplotype A in the heterozygous state. Using genomic DNA from the family members, all exons of the HOXD13 gene with adjacent sequences of exon–intron borders were amplified by PCR as Identification of a nonsense mutation in the HOXD13 gene described previously.24 The amplified PCR products were directly sequenced On the basis of the results of haplotype analysis, we postulated that in an ABI Prism 310 Automated Sequencer, using the ABI Prism Big Dye affected individuals might carry a mutation in the HOXD13 gene with Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, an incomplete penetrance. We next performed direct sequencing Foster City, CA, USA). analysis of the HOXD13 gene of this family. Affected individuals, The mutation c.742C4TintheHOXD13 gene generates a new restriction who are homozygous for haplotype A, carry a homozygous nonsense site for the endonuclease BfaI. A part of exon 1 and intron 1 of the HOXD13 mutation c.742C4T (p.Q248X) in exon 1 of the HOXD13 gene gene was PCR amplified using Platinum Taq DNA Polymerase High Fidelity (Figure 4a). Affected and some unaffected individuals, who are (Invitrogen, Carlsbad, CA, USA) and the following primers: forward (5¢- GTGCCCGGCTATATCGACAT-3¢)andreverse(5¢-AGGCACAACTCCCACT heterozygous for haplotype A, carry the mutation heterozygously. CCCAAGTA-3¢). The amplification conditions were 94 1C for 2 min, followed Unaffected individuals without haplotype A do not carry the muta- by 35 cycles of 94 1C for 30 s, 61 1Cfor30sand681C for 30 s, with a final tion. Restriction enzyme digestion analysis confirmed the result, and extension at 68 1C for 7 min. The amplified PCR products, 280 bp in size, were excluded the mutation from 100 population-matched unaffected purified with the Rapid PCR Purification System (Marligen, Ljamsville, MD, control individuals (Figure 4b; data not shown). USA), digested with BfaIat371C for 3 h and analyzed on 2.0% agarose gels. DISCUSSION RESULTS SPD is a rare genetic malformation of the limb. SPD is clinically Patients heterogeneous, and in addition to the typical clinical presentation of We studied a large consanguineous family from Pakistan with SPD. syndactyly involving the 3/4 fingers and 4/5 toes and digital duplica- The inheritance pattern could be explained either as autosomal tion within the syndactylous web, many patients show a wide range recessive or as autosomal dominant with incomplete penetrance of other clinical manifestations including clinodactyly, camptodactyly, (Figure 1). The clinical presentation among the family members was brachydactyly, duplicated metatarsals in different digits, broad hallux, highly variable. Of 16 affected individuals analyzed, the clinical extra phalangeal creases, widely spaced first and second toes and features of 10 members showed a typical SPD1 phenotype, consisting pre/postaxial digital duplication.4 Variations in clinical presentation of syndactyly between the 3/4 fingers with digital duplication, widely occur not only within different families but also within members of spaced first and second toes, syndactyly involving toes 2/3/4/5 or the same family.4 Atypical clinical features have been more commonly

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Figure 1 Pedigree of a Pakistani family with synpolydactyly consistent with an autosomal dominant pattern with incomplete penetrance. Individuals with and carry the mutation but are not affected. Individuals (a–i) are homozygous for the mutation and individuals (j–l) are heterozygous for the mutation. A full color version of this ﬁgure is available at the Journal of Human Genetics journal online.

Figure 2 Homozygous affected individuals from the Pakistani family with synpolydactyly showed variable and severe phenotypes including syndactyly between the 3/4 fingers with digital duplication, widely spaced first and second toes, syndactyly involving toes 2/3/4/5 or 3/4/5, and camptodactyly of the big toe and brachydactyly, which mainly affected the second toe. A total of 50% of the individuals carrying the heterozygous mutation had the disease, which was characterized by a milder phenotype than those with the homozygous mutation and involved only the feet, consisting mainly of webbing of the fourth and the fifth digits or the second and third digits. In addition, few of the patients had unilateral broad big toes. Individuals (a–i) are homozygous for the mutation and individuals (j–l) are heterozygous for the mutation. A full color version of this figure is available at the Journal of Human Genetics journal online. reported in the setting of deletion and missense mutations than with additional polyalanine repeats are predicted to destabilize the normal polyalanine repeat expansions.4,17–22 protein conformation, leading to its aggregation. This impedes the The role of HOXD13 during embryogenesis is crucial in limb protein translocation from the cytoplasm to the nucleus, in which it development.13,25 It is postulated that the pathophysiology of the functions as a transcription factor.5,26 The mutant proteins also show a abnormal HOXD13 gene is such that mutant HOXD13 proteins with dominant-negative activity against the wild-type protein.6 By contrast,

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Figure 3 Non-parametric linkage analysis reveals a maximum logarithm of odds (LOD) score on chromosome 2q22.3–34 (Z¼3.15). This region contains the HOXD13 gene, which is the cause of synpolydactyly. NPL, non-parametric linkage analysis; HHRR, haplotype-based haplotype relative risk. A full color version of this ﬁgure is available at the Journal of Human Genetics journal online.

Figure 4 (a) Affected individuals, who are homozygous for the haplotype A, carry a homozygous nonsense mutation c.742C4T (p.Q248X) in exon 1 of the HOXD13 gene. Affected and unaffected individuals are heterozygous for haplotype A. (b) The mutation c.742C4TintheHOXD13 gene generates a restriction site for the endonuclease enzyme, thus leading to the digestion of a PCR product from the mutant allele into two fragments of 144 and 136 bp in size. The PCR fragment from the wild-type allele is 280 bp and is not digested. The asterisks represent heterozygotes. A full color version of this ﬁgure is availableattheJournal of Human Genetics journal online.

it was recently demonstrated that the consequence of a missense SPD were homozygous for the mutation, whereas 12 individuals were mutation in the HOXD13 gene was likely to be 50% reduction in heterozygous for the mutation, among which 6 were affected and protein levels, although the underlying mechanism remains unclear.21 showed a mild SPD phenotype. Here, we studied a family from Pakistan with clinically hetero- It is well known that the size of the polyalanine expansion repeats in geneous SPD. Because of the high rate of consanguinity, the inheritance HOXD13 correlates with the severity of the disease. Individuals with pattern was most likely autosomal recessive, while there was also the short repeats (that is, seven or less repeats) are associated with lower possibility of autosomal dominant inheritance with incomplete pene- penetrance and expressivity, whereas those with longer repeats (+10 or trance. We initially performed parametric linkage analysis, which was more) are associated with higher penetrance and more severe dis- non-revealing. We then performed non-parametric linkage analysis ease.27 Moreover, individuals who are homozygous for the polyalanine and identiﬁed linkage to a region on chromosome 2 containing the repeat expansion show a more severe phenotype than those who are HOXD13 gene, under an autosomal dominant inheritance model. heterozygous within members of the same family.24,25,28 Much less is Direct sequencing analysis led to the identiﬁcation of a nonsense known about HOXD13 mutations other than those associated with mutation Q248X in the HOXD13 gene. All 10 individuals with severe polyalanine repeat expansions, because very few families were reported

Journal of Human Genetics A nonsense mutation in the HOXD13 gene MKurbanet al 705 with missense or deletion mutations, and none previously with the mutant protein may be variable between affected individuals, nonsense mutations. The few reports of families with in-frame leading to phenotypic variability in heterozygous mutation carriers deletions and missense mutations presented affected individuals from mild SPD to clinically normal appearance. with only heterozygous mutations and complete penetrance of the In conclusion, our results expand the spectrum of mutations in the disease,17–22 though frameshift mutations could be associated with HOXD13 gene and enhance our understanding of the molecular basis incomplete penetrace.18 The phenotype was generally mild and the of SPD1. clinical features were not consistent with the typical features known for SPD. The mild and atypical phenotypes in non-polyalanine repeat CONFLICT OF INTEREST expansion mutations suggested considerable residual activity of the The authors declare no conflict of interest. HOXD13 gene, which implicated haploinsufficiency rather than a dominant-negative mechanism. Functional studies on a missense ACKNOWLEDGEMENTS mutation designated G220V in the HOXD13 gene in a Greek family We gratefully acknowledge the families for having participated in this study. with SPD showed that haploinsufficiency is likely to be the mechanism We also thank Helen Lam for expert technical assistance. This study was leading to the phenotype, although aggregation of the mutant protein supported in part by the NIH grant from USPHS, NIH/NIAMS RO1 AR44924 within the cytoplasm still occurred, but to a lesser extent than that (to AMC). YS is a recipient of a Research Career Development Award from observed in polyalanine expansions.21 the Dermatology Foundation. In the Pakistani family presented here, we observed highly variable phenotypes among affected individuals. All affected individuals who were homozygous for the mutation had very severe phenotypes. In 1 Ranum, L. P. & Day, J. W. Dominantly inherited, non-coding microsatellite expansion contrast, affected individuals carrying the heterozygous mutation had disorders. Curr. Opin. Genet. Dev. 12, 266–271 (2002). mild and more atypical features. Interestingly, 50% of individuals 2 Albrecht, A. & Mundlos, S. The other trinucleotide repeat polyalanine expansion heterozygous for the mutation were clinically unaffected, suggesting disorders. Curr. Opin. Genet. Dev. 15, 285–293 (2005). 3 Cummings, C. J. & Zoghbi, H Y. Fourteen and counting, unraveling trinucleotide repeat incomplete penetrance of SPD in the setting of nonsense mutations in diseases. Hum. Mol. Genet. 6, 909–916 (2000). HOXD13. 4 Malik, S. & Grzeschik, K. H. Synpolydactyly, clinical and molecular advances. Clin. Genet. 73, 113–120 (2008). It is likely that aberrant HOXD13 transcripts with the nonsense 5 Albrecht, A. N., Kornak, U., Bo¨ddrich, A., Su¨ring, K., Robinson, P. N., Stiege, A. C. mutation Q248X undergo nonsense-mediated mRNA decay, and thus et al. A molecular pathogenesis for transcription factor associated poly-alanine tract no protein will be synthesized from the mutant allele, explaining why expansions. Hum. Mol. Genet. 13, 2351–2359 (2004). 6 Brown, L. Y. & Brown, S. A. Alanine tracts, the expanding story of human illness and affected individuals homozygous for the mutation show a severe SPD trinucleotide repeats. Trends Genet. 20, 51–58 (2004). phenotype. Likewise, the heterozygous mutation is predicted to result 7 Krumlauf, R. Hox genes in vertebrate development. Cell 78, 191–201 (1994). in production of 50% of the protein, leading to either a mild SPD 8 Favier, P. & Dolle, B. Developmental functions of mammalian Hox genes. 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